The invention relates to random nucleic acid mutagenesis using exoxe2x88x92 DNA polymerases.
PCR-based random mutagenesis is widely used for elucidating structure-function relationships of proteins, and for improving protein function (e.g., directed protein evolution) (Cadwell, R. C. and Joyce, G. F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28-33; Leung, D. W., Chen, E., and Goeddel, D. V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11-15). The procedure involves amplifying a gene or portion of a gene under mutagenic conditions, cloning the PCR fragments, and then screening the resulting library for novel mutations that affect protein activity (Melnikov, A. and Youngman, P. J. 1999. Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus. Nucleic Acids Res. 27:1056-1062; Wan, L., Twitchett, M. B., Eltis, L. D., Mauk. A. G., and Smith, M. 1998. In vitro evolution of horse heart myoglobin to increase peroxidase activity. Proc. Natl. Acad. Sci. U.S.A. 95:12825-12831; You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83). Mutations are deliberately introduced during PCR through the use of error-prone DNA polymerases and reaction conditions. To analyze structure-function relationships, mutation rates of 1 mutation per gene are desired to assess the contribution of individual amino acids to protein function (Vartanian, J. P., Henry, M., and Wain-Hobson, S. 1996. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acids Res. 24:2627-2631). For directed evolution, mutagenesis rates of 2 to 7 mutations per gene are considered the most effective for creating mutant libraries and isolating proteins with enhanced activities (Cherry, J. R. Lamsa, M. H., Schneider, P., Vind, J., Svendsen, A., Jones, A., and Pedersen, A.H. 1999. Directed evolution of a fungal peroxidase. Nat. Biotechnol. 17:379-384; Shafikhani, S., Siegel, R. A., Ferrari, E., and Schellenberger, V. 1997. Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. BioTechniques 23:304-310; Wan, L., Twitchett, M. B., Eltis, L. D., Mauk. A. G., and Smith, M. 1998. In vitro evolution of horse heart myoglobin to increase peroxidase activity. Proc. Natl. Acad. Sci. U.S.A. 95:12825-12831; You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83). Mutation rates greater than 7 mutations per gene typically result in loss of protein activity, although proteins with improved activities have been successfully isolated from highly mutagenized libraries exhibiting up to 20 mutations per gene (Daugherty, P. S., Chen, G., Iverson, B. L., and Georgiou, G. 2000. Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies. Proc. Natl. Acad. Sci. U.S.A. 97:2029-2034).
Conventional methods employ Taq DNA polymerase, as it lacks proofreading activity and is inherently error prone. To achieve useful mutation frequencies, the error rate of Taq (1 mutation per xcx9c125,000 bases (Cline, J., Braman, J. C. and Hogrefe, H. H. 1996. PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24:3546-3551) is further increased by employing PCR reaction buffers that contain Mn2+ and/or unbalanced nucleotide concentrations (Cadwell, R. C. and Joyce, G. F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28-33; Leung, D. W., Chen, E., and Goeddel, D. V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11-15). In the presence of 7 mM MgCl2, 0.5 mM MnCl2, 1 mM dCTP and TTP, and 0.2 mM dGTP and dATP, Taq incorporates 4.9 to 6.6 mutations per kb per PCR (Cadwell, R. C. and Joyce, G. F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28-33; Shafikhani, S., Siegel, R. A., Ferrari, E., and Schellenberger, V. 1997. Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. BioTechniques 23:304-310). Under these conditions, mutational bias is regarded as minimal or skewed to favor mutations at AT base pairs. Lower mutation frequencies can be obtained by reducing MnCl2 concentration (1-2 mutations per kb), while higher mutation frequencies ( greater than 6 mutations per kb) are achieved by performing consecutive PCRs or by selectively increasing dGTP concentration (Melnikov, A. and Youngman, P. J. 1999. Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus. Nucleic Acids Res. 27:1056-1062; Nishiya, Y. and Imanaka, T. 1994. Alteration of substrate specificity and optimum pH of sarcosine oxidase by random and site-directed mutagenesis. Appl. Env. Microbiol. 60:4213-4215; You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83).
Although widely used, Taq-based methods exhibit significant drawbacks that limit the utility of PCR random mutagenesis methods. First, amplification under mutagenic conditions (Mn2+, unbalanced nucleotide pools) reduces the activity of Taq and limits random mutagenesis to DNA sequences less than 1-kb in length (Leung, D. W., Chen, E., and Goeddel, D. V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11-15; Stemmer, W. P. 1994. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91:10747-10751). Second, PCR products are amplified in lower yield using mutagenic reaction conditions (Vartanian, J. P., Henry, M., and Wain-Hobson, S. 1996. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acids Res. 24:2627-2631), which can reduce cloning efficiency and library size. Third, preparing and using multiple buffers (varying MnCl2 and dNTP concentrations) to construct a series of libraries with different mutation frequencies is time-consuming and can produce variable results. Finally, altering nucleotide ratios to achieve high mutation frequencies ( greater than 6 mutations per kb) can lead to strong bias in the types of mutations produced. For example, selectively increasing dGTP concentration favors ATxe2x86x92GC transitions, which accounted for 70% of all mutations in one study (You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83).
There is a need in the art for random mutagenesis of nucleic acid longer than 1 kb. There is also a need to improve the yield of the final mutated product to facilitate subsequent cloning of the product. There is further a need for a novel error prone DNA polymerase which minimizes mutation bias or produces a different mutational bias than a given polymerase produces. Finally, there is a need for a simplified PCR mutagenesis conditions to achieve various mutation frequencies.
The invention is related to novel compositions and methods for nucleic acid mutagenesis.
The invention provides a composition for PCR mutagenesis comprising an archaeal exoxe2x88x92 DNA polymerase which substantially lacks 3xe2x80x2 to 5xe2x80x2 exonuclease activity, and PCR enhancing factor.
In a preferred embodiment, the archaeal exoxe2x88x92 DNA polymerase is selected from the group consisting of: exoxe2x88x92Tli DNA polymerase, exoxe2x88x92 Pfu DNA polymerase, exoxe2x88x92 KOD DNA polymerase, exoxe2x88x92 JDF-3 DNA polymerase, and exoxe2x88x92PGB-D DNA polymerase.
The invention also provides a composition comprising an archaeal exoxe2x88x92 DNA polymerase, PCR enhancing factor, and one or more DNA polymerases selected from the group consisting of Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exoxe2x88x92Tli DNA polymerase, exoxe2x88x92 Pfu DNA polymerase, exoxe2x88x92 Tma DNA polymerase, exoxe2x88x92 KOD DNA polymerase, exoxe2x88x92 JDF-3 DNA polymerase, and exoxe2x88x92PGB-D DNA polymerase, wherein said one or more DNA polymerases are different from said archaeal DNA polymerase.
Preferably the compositions mentioned above herein further comprise a PCR buffer useful for generating a mutated amplified product at a given mutation frequency.
More preferably, the PCR buffer lacks Mn2+.
The compositions mentioned above-herein may further comprise equivalent molar amounts of dATP, dTTP, dGTP, and dCTP.
The invention provides a kit for PCR mutagenesis comprising an archaeal exoxe2x88x92 DNA polymerase, PCR enhancing factor, and packaging means therefore.
The invention further provides the above-mentioned kit, further comprising one or more polymerases selected from a group consisting of Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exoxe2x88x92Tli DNA polymerase, exoxe2x88x92 Pfu DNA polymerase, exoxe2x88x92Tli DNA polymerase, exoxe2x88x92 Tma DNA polymerase, exoxe2x88x92 KOD DNA polymerase, exoxe2x88x92 JDF-3 DNA polymerase, and exoxe2x88x92PGB-D DNA polymerase, wherein said one or more DNA polymerases are different from said archaeal DNA polymerase.
The kits mentioned above-herein may further comprise a PCR buffer useful for generating a mutated amplified product at a given mutation frequency.
Preferably, the PCR buffer in the above mentioned kits lacks Mn2+.
More preferably, the kits further comprise equivalent molar amounts of dATP, dTTP, dGTP, and dCTP.
The invention provides a method of PCR amplification for mutagenesis comprising incubating a reaction mixture comprising a nucleic acid template, at least two PCR primers, an archaeal exoxe2x88x92 DNA polymerase, and PCR enhancing factor under conditions which permit amplification of said nucleic acid template by said archaeal exoxe2x88x92 DNA polymerase to produce a mutated amplified product.
Preferably, the archaeal exoxe2x88x92 DNA polymerase is selected from the group consisting of: Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exoxe2x88x92Tli DNA polymerase, exoxe2x88x92 Pfu DNA polymerase, exoxe2x88x92 Tma DNA polymerase, exoxe2x88x92 KOD DNA polymerase, exoxe2x88x92 JDF-3 DNA polymerase, and exoxe2x88x92PGB-D DNA polymerase.
The above-mentioned method, may further comprise incubating one or more exoxe2x88x92 DNA polymerases selected from a group consisting of: Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exoxe2x88x92Tli DNA polymerase, exoxe2x88x92 Pfu DNA polymerase, exoxe2x88x92Tli DNA polymerase, exoxe2x88x92 Tma DNA polymerase, exoxe2x88x92 KOD DNA polymerase, exoxe2x88x92 JDF-3 DNA polymerase, and exoxe2x88x92PGB-D DNA polymerase in said reaction mixture, wherein said one or more DNA polymerases are different from said archaeal DNA polymerase.
Preferably, said incubating step is performed in a PCR reaction buffer lacking Mn2+.
Also preferably, said incubating step may further comprise incubating equivalent molar amounts of dATP, dTTP, dGTP, and dCTP.
Still preferably, said incubating step may generate said mutated amplified product at a given mutation frequency using a given amount of said nucleic acid template.
The method useful to the invention may comprise a first said incubating step which generates a first said mutated amplified product at a first given frequency using a first selected amount of said nucleic acid template, and a second said incubating step which generates a second said mutated amplified product at a second given frequency using a second selected amount of said nucleic acid template, wherein said first incubating step and second incubating step comprise a single buffer composition.
The method of the invention may further comprise subsequently repeating one or more additional said incubating step using a portion of or the total amplified product of a preceding incubating as template for a subsequent incubating step.
Preferably, the mutation frequency generated by the incubating step is proportional to the amount of said nucleic acid template.
The incubating step of the subject invention may comprise 1 pg to 1 xcexcg of said nucleic acid template, which may produce said mutated amplified product from said nucleic acid template at a mutation frequency of 1,000 to 16,000 mutations or more per 106 base pair.
The incubating step of the subject invention may comprise 10-100 ng of said nucleic acid template, which may produce said mutated amplified product at a mutation frequency of 1,000 to 3,000 mutations per 106 base pair.
The incubating step of the subject invention may comprise 10 pg to 10 ng of said nucleic acid template, which may produce said mutated amplified product at a mutation frequency of 3,000 to 7,000 mutations per 106 base pair.
The incubating step of the subject invention may comprise 10 pg to 10 ng of said nucleic acid template, which may produce said mutated amplified product at a mutation frequency of 7,000 to 16,000 or more mutations per 106 base pair.
According to the instant invention, one or more additional said incubating steps may be repeated subsequently using a portion of or the total amplified product of a preceding incubating as template for a subsequent incubating to generate a mutation frequency of 7,000 to 16,000 or more mutations per 106 base pair.
The nucleic acid template of the instant invention may be 0.1 kb to 10 kb in length.
The method of the instant invention may produce an amplified product at a yield of 0.5-10 xcexcg.